Mindfully.org  

Home | Air | Energy | Farm | Food | Genetic Engineering | Health | Industry | Nuclear | Pesticides | Plastic
Political | Sustainability | Technology | Water
Perc removal


Genetically Engineered Crops

A Threat to Soil Fertility? 

PSRAST* 21mar01

*Physicians and Scientists for Responsible Application of Science and Technology

For reasons explained elsewhere, the list of authors can not presently be disclosed. So it is presently published in the name of PSRAST.

Among the contributors to this paper have been:

Editor: Jaan Suurkula M.D.


The decisive role of Soil Ecology for fertility

Recent research has demonstrated the great importance of soil organisms for the fertility of the soil. In one gram of productive soil there is a complex web that can exceed over 100 million micro-organisms that may represent over 1000 species. The main components are bacteria, fungi, algae, protozoa's, nematodes, earthworms, and insects. Out of these, bacteria and fungi constitute about 80%, the proportions of these two depending on soil type. There is a complex ecological interdependence between all soil organisms. Together they are responsible for the cycle of decomposing and restructuring organic material so that it will be accessible to growing plants. Also it is responsible for the nitrogen - and water-retaining properties as well as for other factors of great importance for soil fertility. "Without the soil foodweb, plants would not obtain the nutrients necessary for growth, and the above ground foodweb would not long continue" according to Nannipieri, P. et al. (1990).

The knowledge about soil microecology is still incomplete but enough is known to conclude that it is of decisive importance for soil fertility. Ingham. has established measurable criteria for predicting soil fertility on the basis of the microbial conditions in the soil, see Ingham, E. (1998).

Naseby D.C and Lynch J.M. (1998) wrote "Because of the importance of soil biota in mineralization and immobilization of nutrients, physical and biochemical degradation of organic matter, biological control of plant pests, and as food sources for other organisms, it is crucial to evaluate the potential impacts of transgenic plants on soil ecosystems".

Potential effects of Genetically Engineered crops on soil microbes

In genetic engineering a package of genes are inserted into the recipient organism. In addition to the desired property gene, a number of other genes have to be added to ensure successful insertion.

Amongst the potentially problematic genes inserted in plants, those that help overcome the barriers against the introduction of foreign genes are of particular interest in the context of soil ecology. They function as vectors for successful insertion and prevent rejection of inserted foreign genes. These vector packages are chimaeric combinations of genes commonly from pathogenic bacteria and viruses and from transposons.

Ho, M.W. & Tappeser, B. (1997) have forwarded the hypothesis that the vector genes in GE crops may promote horizontal transfer of genetic material between unrelated bacterial species. They warned that the result may be new human pathogenic bacteria.

This was further developed by Ho, M.W. et al. (1998). They refer to extensive experimental evidence showing the existence of different mechanisms for gene transfer not only between related bacteria but also between bacteria of different species, as well as between bacteria and fungi and between bacteria and higher organisms, including mammals. Also transfer of genes from transgenic plants to soil bacteria and soil fungi has been reported. They warn that the vector genes may be transferred to soil bacteria and soil fungi and considerably contribute to increased horizontal transfer. They suggest that this may have contributed to the emergence of new human pathogenic bacteria during the last 10-15 years, some of which have been very harmful.

Hypothesis

We want now to extend the hypothesis of Ho, M.W. & Tappeser, B. (1997) and Ho, M.W. et al. (1998) to Soil Ecology. We suggest horizontal transfer promoted by vector genes from the GE plants may cause genetic disturbances in soil micro-organisms. If this mechanism works to a significant extent this might in the worst case result in alterations in soil microbial ecology resulting in decreased soil fertility.

As important parts of the reasoning below is based on the hypothesis of Ho et al (1998), relevant parts of the text have been taken from there with permission.

Evidence in support of the hypothesis

Inductive

1. Direct experimental evidence of horizontal gene transfer, some between phylogenetically distant species, has been obtained in all natural environments as well as in the gastrointestinal tract. These were all accomplished using artificially constructed vectors.
2. DNA released from dead cells (as well as live cells) are not readily broken down in the general environment, nor in the gastrointestinal tract, where they may retain the ability to transform other bacteria.

Deductive

1. Genetic engineering is based on facilitating horizontal gene transfer between distant species by constructing vectors that break down species barriers.
2. The artificial vectors constructed for genetic engineering are chimaeric combinations of viral pathogens and other invasive genetic elements that can generate new cross-species pathogens.
3. The artificial vectors constructed for genetic engineering are inherently unstable and prone to recombination, thereby enhancing horizontal gene transfer and recombination.
4. Shuttle vectors made by genetic engineering are essentially unstoppable, as they contain signals for transfer and replication in different species; and helper functions for mobilization and transfer can be supplied by viruses, plasmids and transposons which occur naturally in bacteria in all environments.

Inductive evidence

1. Transfers have occurred from bacteria to higher plants and vice versa.

The best known example is the direct demonstration of transfer between the soil bacterium, Agrobacterium and plants. In a process bearing a strong resemblance to conjugation between bacteria, the tumour (T) segment of the tumour-inducing (Ti) bacterial plasmid is transferred and incorporated into the plant genome (Kado, C.I., 1993; Stachel, S.C., Timmerman, B. & Zambryski, P., 1986). However, it must be noted that nearly all cases of directly demonstrated horizontal gene transfer, especially those involving phylogenetically distant species, made use of already modified hybrid shuttle vectors that can transfer between species and replicate in both. These shuttle vectors possess signals for replication (origins of replication) in more than one species as well as the signal for DNA transfer (origin of transfer), and are hence much more likely to be successful in horizontal transfer than unmodified plasmids found naturally. The Ti plasmid is, indeed, the basis of a gene transfer vector system widely used for genetically engineering crop-plants.

Cross-Kingdom horizontal gene transfer by conjugation has also been demonstrated between bacteria and yeast using shuttle vectors derived from broad host-range promiscuous plasmid which are already transferable between many bacterial species (Heinemanne, J.A. & Sprague, G.R., Jr., 1989; Sikorski R.S. & Hieter, P., 1989). Such vectors can even substitute for the Ti plasmid in transferring genes from Agrobacterium to plants (Buchanan-Wollaston, V. Passiatore, J.E. & Cannon, F., 1987). Recent evidence documented the direct transfer of transgenes and marker genes from transgenic plants to soil fungi (Hoffman, T., Golz, C. & Schieder, O., 1994) and soil bacteria (Schluter, K., Futterer, J. & Potrykus, I., 1995), indicating that secondary, unintended gene transfers can occur from genetically engineered crop-plants which are now released commercially into the environment. Despite the title of their publication, Schluter, K. et al. (1995) actually observed a high "optimal" gene transfer frequency of 6.2 x 10-2 in the laboratory, from which they "calculated" a frequency of 2.0 x 10-17 under extrapolated "natural conditions".

Transformation (pieces of genetic material taken up into the cell from the environment) may be a major route of horizontal gene transfer: frequencies obtained under different environmental conditions, using artificial vectors and markers are found to be generally quite high, many ranging between 10-2 to 10-5 transformant per viable cell. In fact, transformation frequencies are often higher under natural conditions than in the laboratory.

In a recent study in soil microcosms, activity due to earthworms was found to significantly enhance gene transfer between spatially separated bacterial species inoculated into the soil (Daane, L.L., Molina, J.A.E. & Sadowsky, M.J., 1997). A special form of bi-directional transformation by cell contact and fusion is now known to be widespread (Yin, X. & Stotzky, G., 1997).

Presence of naked DNA in the soil, the requirement for frequent transformation, is considerable.

2. DNA released from plant cells are not readily broken down in the general environment

Genes carried by vectors as naked DNA as well as chromosomal DNA can survive indefinitely, especially when adsorbed to solid particles in all environment; on surfaces of water, as sediment, in the soil, where they are efficiently taken up by other microbes (reviewed in Crecchio, C. & Stotzky, G., 1996; Stotzky G, 2000; Jager, M.J. & Tappeser, B., 1995). Although DNA is rapidly broken down in waste water, adsorption to solid particles in the sludge, which happens very quickly, will stabilize it and prolong its transforming capacity. In addition, DNA is now found to persist for long periods in the laboratory clinging to many different surfaces. Transformation by the uptake of DNA is a major route of horizontal gene transfer in the environment. DNA is not only released when cells die, but are actively secreted by living cells, and even fragments of genes can have significant effects when transferred.

DNA is not only released into the environment when the cells die, but is actively excreted by living cells during growth. Some species export DNA wrapped in membrane-bound vesicles. The DNA in a culture slime can be more than 40% of the dry weight. Thus, the environment is extremely rich in DNA. Fresh water contains between 0.5 to 7.8mg per liter; while freshwater sediment has an upper concentration of 1mg per gram. Although enzymes breaking down DNA (deoxyribonucleases, DNases) are found in the environment, DNA is protected from degradation by adsorbing to detritis, fulvic acid, and in particular, clay and sand particles. Adsorbed DNA is equally efficient in transforming cells. Thus, the half-lives of DNA in soil is 9.1 hours for loamy sand soil, 15.1 h for silty clay soil and 28.2 h for clay soil. While half-lives (time for half of the DNA to be broken down) in waste water are typically fractions of an hour, those in freshwater and marine water are 3 to 5 hours, with high values of 45 to 83 h on the ocean surface, and extremely high values of 140 and 235 hours for the marine sediment (Lorenz, M.G. & Wackernagel, W., 1994).

Adsorption of DNA to solid particles is a very rapid process, which means that DNA released into the environment can survive indefinitely and maintain its potential to transform other organisms.

Deductive Evidence

1. Genetic engineering is based on facilitating horizontal gene transfer between distant species by constructing vectors that break down species barriers.

2. The artificial vectors constructed for genetic engineering are chimaeric combinations of viral pathogens and other invasive genetic elements that can generate new cross-species viral pathogens.

3. The artificial vectors constructed for genetic engineering are inherently unstable and prone to recombination, thereby enhancing horizontal gene transfer and recombination.

4. Shuttle vectors made by genetic engineering are essentially unstoppable, as they contain signals for transfer and replication in different species; and helper functions for mobilization and transfer can be supplied by viruses, plasmids and transposons which occur naturally in bacteria in all environments.

One main contributing factor to the recent increase in the scope and frequency of horizontal gene transfer may be the deliberate acts of genetic engineers to break down species barriers. They do so by constructing a range of chimaeric vectors for cloning and transferring genes. These artificial vectors have the following important characteristics that enhance horizontal gene transfer.

Although different classes of vectors are distinguishable on the basis of the main framework sequence, practically everyone of them is chimaeric. Important chimaeric vectors are the shuttle vectors which enable genes to be cloned (multiplied) in E. coli and transferred (transfected) into unrelated species in every Kingdom. Similarly, vectors used in manipulating plants and animals typically contain sequences from a range of plant and animal viral pathogens, as well as antibiotic resistance genes, often originating from promiscuous resistance plasmids and transposons. Phage vectors and phasmid vectors (hybrid of phage and plasmid) are also extensively used, and may have special relevance for the evolution of pathogenicity islands in bacterial pathogens.

Thus, genetic engineering biotechnology has effectively opened up highways for horizontal gene transfer and recombination, where previously, there was only restricted access through narrow, tortuous footpaths. Nielsen et al (1998) wrote:

" Transfer frequencies should not be confounded with the likelihood of environmental implications, since the frequency of [horizontal gene transfer] is probably only marginally important compared with the selective force acting on the outcome. Attention should therefore be focused on enhancing the understanding of selection processes in natural environments. Only an accurate understanding of these selective events will allow the prediction of possible consequences of novel genes following their introduction into open environments".

We review further circumstantial evidence that artificial gene transfer vectors increase the scope and frequency of horizontal gene transfer.

Circumstantial evidence that artificial gene transfer vectors increase the scope and frequency of horizontal gene transfer.

It is not easy to transfer genes successfully between species as we have already emphasized, there are barriers to horizontal gene transfers (see Ingham, E.). That is why, apart from transposons which are promiscuous, such events were relatively rare in our evolutionary past.

Horizontal gene transfers have been directly demonstrated between bacteria in the marine environment (Frischer, M.E., Stewart, G.J. & Paul, J.H., 1994; Lebaron, Ph., Batailler, N. & Baleux, B., 1994; Sandaa, R.A. & Enger, Ø., 1994), in the freshwater environment (Ripp, S., Ogunseitan, O.A. & Miller, R.V., 1994) and in the soil (Neilson, J.W., Josephson, K.L., Pepper, I.L. et al., 1994). Again, in all the experiments, horizontal gene transfers were mediated by specially constructed hybrid plasmid vectors, of the sort used in genetic engineering. Horizontal gene transfer occurs preferentially in interfaces between air and water and in the sediment, and especially under nutrient depletion conditions (Goodman, A.E., Marshall, K.C. & Hermansson, M., 1994), thus refuting the claim that nutrient-rich media are necessary to support horizontal gene transfer. Horizontal gene transfer of antibiotic resistances has even been demonstrated in wastewater treatment ponds, the effluent from which is increasingly being used for irrigation in developing countries (Mezrioui, N. & Echab, K., 1995). As pointed out in Ho, M.W. et al. (1998, section 7.1), frequencies of horizontal gene transfer may be greater under natural conditions than in the laboratory (Daane, L.L., Molina, J.A.E. & Sadowsky, M.J., 1997).

Stephenson & Warnes (1996, p.5) wrote, 

"The threat of horizontal gene transfer from recombinant organisms to indigenous ones is ..very real and mechanisms exist whereby, at least theoretically, any genetically engineered trait can be transferred to any prokaryotic organism and many eukaryotic ones.".

A year later, another molecular geneticist, who works on transgenic plants, admitted that, 

"..the potential for horizontal [gene] transfer may be greater than thought previously." (Harding, K., 1996).

Potential agricultural consequences

The great problem in predicting potential outcomes here is that the suggested horizontal transfer mechanism would have very diverse results, impossible to predict. Therefore it appears impossible to exclude, on a theoretical basis, that the effect may result in changes that will not affect soil fertility. Only by multiple experiments will it be possible to get some idea whether this represents a significant risk.

1. One possible complication might come from horizontal transfer of the Bacillus thuringiensis toxin (Bt) gene to soil bacteria. The Bt gene is often used in GE crops to protect from common pests. Stotsky et al have recently shown that Bt toxins from GE crops will, unlike natural Bt toxin not disappear when added to soil, but become rapidly bound to soil particles, and are not broken down by soil microbes.

It is warned that engineered Bt toxins could build up in the soil, killing Bt sensitive soil organisms. In addition, the researchers suspect that a broader range of organisms is likely to be susceptible to the active, engineered toxins, since some organisms lack the enzymes to inactivate it. ( Koskella J and Stotzky G. (1997). Tapp H. and Stotzky G. (1998).

If the Bt gene is taken up by soil bacteria, some might perhaps produce Bt toxins. If this would occur extensively, a significant production of these toxins might ensue, leading to damage to soil organisms. This disturbance might persist for long time, perhaps indefinitely after the cultivation of Bt-gene carrying crops has ceased, depending on the vitality of toxin-producing strains. It cannot be excluded that it might be difficult to eradicate such bacteria.

2. Another scenario might be that gradually, there occurs an accumulation of genetic disturbances most of which are harmless to the soil micro-organisms. But over long time this might lead to a gradual increase of the numbers of altered soil micro-organisms that are unable to uphold the functions required for good soil productivity.

Damage to soil fertility caused by such a slowly growing "genetic chaosis" would not become obvious before extensive damage has occurred, which might take several years. The serious aspect with this kind of complication, if it turns out to be a real outcome, is that it may be very difficult if not impossible to repair as there will be a gigantic reservoir of horizontal transfer promoting vectors in the soil flora. In a region where transgenic crops have been cultivated, this might ultimately lead to irreparable reduction of soil fertility.

3. A third scenario might be that a new soil microorganism variety or species might arise that is able to overgrow or extinguish some essential soil microorganisms so that the ecological balance would be disrupted. If it is hardy it might spread widely through ground water and soil erosion.

Conclusion

The reviewed evidence support the possibility of horizontal transfer of transgenic vector genes from transgenic crops to soil microorganisms. Experimental evidence indicate that such vectors can promote interchange of genetic material between micro-organisms that rarely or never exchange genes.

This might give rise to genetic disturbances that in the worst case might disrupt soil ecology and thereby damage soil fertility. Another potential complication might be the uptake of Bt toxin genes by soil microbes leading to Bt toxin production in the soil which may be harmful to soil organisms.

As it appears conceivable that the transgene vectors may persist in a significant part of soil microorganisms, it cannot be excluded that they will accumulate as long as transgenic crops are cultivated. And it cannot be excluded that they might persist for a significant time or perhaps indefinitely even after cultivation of transgenic crops has ceased.

The presented evidence are indirect and it is impossible to judge, without direct experimental investigation, how probable it is for the proposed mechanism to work in practice to a significant extent and to result in altered soil microorganisms and in disturbed soil ecology to such a degree that it will result in decreased soil fertility.

As:

  1. The suggested mechanism might in the worst case cause irreparable, cumulative and persistent damage to soil fertility
  2. The genes causing the complication might spread uncontrollably
  3. The available evidence supporting this possibility is not insignificant
  4. None of presently grown GE crops are of any significant value to mankind,

     

we find that it is unjustifiable to continue the culture of any transgenic crops until it has been established experimentally beyond reasonable doubt that the proposed mechanism may not result in extensive and persistent disturbances of soil ecology.

Published on March 21, 2001

References

Buchanan-Wollaston, V. Passiatore, J.E. & Cannon, F. (1987). The mob and oriT mobilization functions of a bacterial plasmid promote its transfer to plants. Nature 328: 172-175.

Crecchio, C. & Stotzky, G. (1996). Binding of DNA from Bacillus subtilis on soil humic acids: implications for bacterial transformation in soil. Abst. 8th Meeting. International Humic Substances Society (in press).

Daane, L.L., Molina, J.A.E. & Sadowsky, M.J. (1997). Plasmid transfer between spatially separated donor and recipient bacteria in earthworm-containing soil microcosms. Applied and Environmental Microbiology 63 (2): 679-686.

Frischer, M.E., Stewart, G.J. & Paul, J.H. (1994). Plasmid transfer to indigenous marine bacterial-populations. FEMS Microbiology Ecology 15: 127-135.

Goodman, A.E., Marshall, K.C. & Hermansson, M. (1994). Gene transfer among bacteria under conditions of nutrient depletion in simulated and natural aquatic environments. FEMS Microbiology Ecology 15: 55-60.

Harding, K. (1996). The potential for horizontal gene transfer within the environment. Agro-Food-Industry Hi-Tech. July/August: 31-35.

Heinemanne, J.A. & Sprague, G.R., Jr. (1989). Bacterial conjugative plasmids mobilize DNA transfer between bacteria and yeast. Nature 340: 205-209.

Ho, M.W. & Steinbrecher, R. (1997). Fatal Flaws in Food Safety Assessment. A critique of the joint FAO/WHO Biotechnology and Food Safety Report. Third World Network, Penang.

Ho, M.W. & Tappeser, B. (1997). Potential contributions of horizontal gene transfer to the transboundary movement of living modified organisms resulting from modern biotechnology. In Transboundary Movement of Living Modified Organisms Resulting from Modern Biotechnology: Issues and Opportunities for Policy-Makers (K.J. Mulongoy, ed.) pp.171-193, International Academy of the Environment, Switzerland.

Ho, M.W. et al., "Gene Technology and Gene Ecology of Infectious Diseases" Microbial Ecology in Health and Disease 10: 33-39 1998.

Hoffman, T., Golz, C. & Schieder, O. (1994). Foreign DNA sequences are received by a wild-type strain of Aspergillus niger after co-culture with transgenic higher plants. Current Genetics 27: 70-76.

Höfle, M.G. (1994). Auswirkungen der Freisetzung backerieller Monokulturen auf die naturliche Midroflora aquatischer Okosysteme. In Biologische Sicherheit/Forschung Biotechnologie BMFT vol 3: 795-820

Ingham, E. (1998)". The Soil Foodweb: It's Importance in Ecosystem Health" at http://www.rain.org/~sals/ingham.html).

Jager, M.J. & Tappeser, B. (1995). Risk Assessment and Scientific Knowledge. Current data relating to the survival of GMOs and the persistence of their nucleic acids: Is a new debate on safeguards in genetic engineering required? - considerations from an ecological point of view. Preprint circulated and presented at the TWN-Workshop on Biosafety, April 10, New York. A shortened version appeared in 1996 as Politics and science in risk Assessment. In Coping with Deliberate Release. The Limits of Risk Assessment (A.van Dommelen, ed.), pp.63-72, International Centre for Human and Public Affairs, Tilburg.

Kado, C.I. (1993). Agrobacterium-mediated transfer and stable incorporation of foreign genes in plants. In Bacterial Conjugation (D.B. Clewell, ed.), pp. 243-254, Plenum Press, New York.

Koskella J and Stotzky G. (1997)"Microbial Utilization of Free and Clay-Bound Insecticidal Toxins from Bt and Their Retention of Insecticidal Activity after Incubation with Microbes," Applied and Env. Microbiology, Sept. 1997, p. 3561-3568.

Lebaron, Ph., Batailler, N. & Baleux, B. (1994). Mobilization of a recombinant nonconjugative plasmid at the interface between wastewater and the marine coastal environment. FEMS Microbiology Ecology 15: 61-70.

Lorenz, M.G. & Wackernagel, W. (1994). Bacterial gene transfer by natural genetic transformation in the environment. Microbiological Reviews 58: 563-602.

Mezrioui, N. & Echab, K. (1995). Drug resistance in Salmonella strains isolated from domestic wastewater before and after treatment in stabilization ponds in an arid region (Marrakech, Morocco). World Journal of Microbiology & Biotechnology 11: 287-290.

Nannipieri, P., Grego, S., & Ceccanti, B. (1990). Ecological significance of the biological activity in soil. Soil Biochemistry 6: 293-355.

 Naseby D.C. and Lynch J.M. (1998). Impact of wild-type and genetically modified Pseudomonas fluorescens on soil enzyme activities and microbial population structure in the rhizosphere of pea. Molecular Ecology 7 : 617-625.

Nielsen, K.M., A.M. Bones, K. Smalla, and van Elsas J. D. (1998). Horizontal gene transfer from transgenic plants to terrestrial bacteria - a rare event? FEMS Microbiological Reviews 22: 79-103.

Neilson, J.W., Josephson, K.L., Pepper, I.L., Arnold, R.B., Digiovanni, G.D. & Sinclair, N.A. (1994). Frequency of horizontal gene-transfer of a large catabolic plasmid (PJP4) in soil. Applied Environmental Microbiology 60: 4053-4058.

Old, R.W. & Primrose, S.B. (1994). Principles of Gene Manipulation (5th ed.), Blackwell Science, Oxford.

Ripp, S., Ogunseitan, O.A. & Miller, R.V. (1994). Transduction of a fresh-water microbial community by a new Pseudomonas-aeruginosa generalized transducing phage, UTI. Molecular Ecology 3: 121-126.

Sandaa, R.A. & Enger, Ø. (1994). Transfer in marine sediments of the naturally occurring plasmid pRAS1 encoding multiple antibiotic resistance. Applied and Environmental Microbiology 60: 4243-4238.

Schluter, K., Futterer, J. & Potrykus, I. (1995). Horizontal gene-transfer from a transgenic potato line to a bacterial pathogen (Erwinia-chrysanthem) occurs, if at all, at an extremely low-frequency. Bio/Techology 13: 1094-1098.

Sikorski, R.S. & Hieter, P. (1989). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122: 19-27.

Smalla, K. 1997, personal communication.

Stachel, S.C., Timmerman, B. & Zambryski, P. (1986). Generation of single-stranded T-DNA molecules during the initial stages of T-DNA transfer from Agrobacterium tumefaciens to plant cells. Nature 322: 706-712.

Stephenson, J.R. & Warnes, A. (1996). Release of genetically-modified microorganisms into the environment. J. Chem. Tech. Biotech. 65: 5-16.

Stotzky, G.J. (2000) Persistence and biological activity in soil of insecticidal proteins from Bacillus thuringiensis and of bacterial DNA bound on clays and humic acids. Journal of Environmental Quality 29:691-705.

Tapp H. and Stotzky G. (1998) "Persistence of the Insecticidal Toxin from Bt subsp. Kurstaki in Soil," Soil Biology and Biochemistry, Vol. 30, No. 4, p. 471-476.

Yin, X. & Stotzky, G. (1997). Gene transfer among bacteria in natural environment. Applied Microbiology (in press).

If you have come to this page from an outside location click here to get back to mindfully.org